1. Field of the Invention
The invention relates generally to the detection of antibodies and more specifically to methods for detection of antibodies including a generalized approach applicable to any antibody along with antibody specific assays developed using the approach.
2. Background Information
Antibodies and other polypeptides are widely used in a variety of applications, from use as therapeutic agents to use as reagents in biological assays. The ability to detect and quantify antibodies is a key aspect in many of the applications for which they are currently used. For instance, detection of antibodies and other protein therapeutics in a biological sample is key in medical applications requiring determination of diagnostic and/or prognostic factors. However, determining the pharmacokinetics of a particular antibody can be problematic, especially where the detection is performed against a high background, as in a biological sample.
The need for accurate pharmacokinetic determinations is acute, but has not yet been met by existing methodologies. Dosing schedules and routes of administration for antibodies are developed with many uncertainties and are often refined through pre-clinical and clinical studies where the need for simple and reliable pharmacokinetic assays is acute. The specific biology of a given antibody target can also lead to additional complications from the conventional dose escalation paradigm. For instance, there have been reports of cell free CD20 in the plasma of chronic lymphoid leukemia (CLL) patients (Manshouri et al., Blood, 101(7):2507, 2003). Other reports have suggested that CD20 may be “shaved” from CLL cells consequent to rituximab treatment (Williams et al., J. Immunol., 177(10):7435, 2006), which may or may not affect the detection of free rituximab (Beum et al., J. Immunol. Methods, 289(1-2):97, 2004). Accordingly, detection of noncomplexed antibody is a critical factor for reliable prognostics and diagnostics.
To date, several approaches have been used to study the pharmacokinetics of these treatments, in part to determine diagnostic and prognostic factors, such as progression of cancer. One approach entails making the target molecule recombinantly for use in assays such as sandwich enzyme-linked immunosorbent assays (ELISAs) (Tan et al., Clin. Cancer Res., 12(21):6517, 2006). However, it may not always be possible to generate the recognized portion of the target molecule and it is expensive and cumbersome to generate large amounts of recombinant protein.
An alternative approach involves generating peptides designed from the target antigen sequence, which has been met with limited success (Blasco et al., J. Immunol. Methods, 325(1-2):127, 2007). Another alternative is to express the target molecule on a cell line by transfection, using flow cytometry to assess the binding of the desired antibody (Rebello et al., J. Immunol. Methods, 260(1-2):285, 2002). This method has been used for the antibody therapeutic alemtuzumab (anti-CD52), but is difficult to develop, requires skilled personnel to execute, and has limited sensitivity (Hale et al., Blood, 104(4):948, 2004).
In yet another approach, ELISA assays have been developed that use antibodies specific for the therapeutic antibody (Montagna et al., Int. J. Immunopathol., Pharmacol. 20(2):363, 2007). The antibodies used for this purpose are either anti-idiotypic (Maloney et al., Blood, 90(6):2188, 1997) or specific for residual non-human sequences of the therapeutic antibody, as was the case with alemtuzumab (Mani et al., Leuk. Res., 28(12):1255, 2004). However, each of these approaches is technically demanding and has limited sensitivity when used in biologic samples because of high background. The latter approach in particular will not work with fully humanized antibodies.
In light of the limitations of developing assays for detection of specific antibodies or other peptide therapeutics, a need exists for a single, inexpensive generalized approach of assay development which may be used for development of highly specific assays for a variety of antibodies or other recombinant proteins. Such assays will not only be useful in pharmacokinetic studies but, due to their high sensitivities, can also be applied to diagnosis and prognosis of diseases associated with the presence or absence of the targeted molecules in a biological sample.